Gel filtration of mouse and rat pituitary extracts on guanidine HC1 columns reveals the presence of three classes of molecules with adrenocorticotrophic hormone activity (ACTH) (activity determined by bioassay and radioimmunoassay). Extracts of mouse pituitary tumor cells (AtT-20/D16v line) contain the same size classes of molecules. The highest molecular weight form is the size of growth hormone (25,000), the second class is about 8,000 in molecular weight, and the third class is the size of porcine ACTH (5,000 in molecular weight). These classes of molecules are being purifed from tumor cell cultures for structural studies and for the preparation of antisera to use for immunoprecipitation and radioimmunoassays of the different forms of ACTH. These methods will be used for isolating and identifying radioactive products made: (1) in pulse chase experiments with tumor cells and (2) in experiments in which messenger RNA from tumor cells is translated in cell free systems. A membrane bound ribosome fraction has been isolated from the tumor cells that has ACTH tightly bound to it (presumably in the form of nascent chains). Primary monolayer cultures have been prepared from mouse pituitary that secrete ACTH. The effects of steroids, hypothalamic extracts and other agents on secretion of ACTH by these cultures and by tumor cells are being investigated.